Sampling and detection of African swine fever virus within a feed manufacturing and swine production system

Abstract Transmission of biological hazards capable of causing disease in livestock can occur through a wide variety of direct and indirect routes. In swine production, there are a large number of possible routes of exposure of a pathogen into a susceptible population. African swine fever virus (ASFV) has been a significant challenge for Southeast Asia since first detected in China in 2018 and has spread through many countries within the region. In order to understand potential transmission pathways within an ASFV endemic region, a diagnostic investigation was performed to determine the level of contamination on a wide variety of surface types within a live animal production, feed manufacturing, and feed distribution system located in Vietnam. All diagnostic testing was performed locally by the production system's internal diagnostic laboratory using real‐time polymerase chain reaction (rt‐PCR) analysis. Early in the diagnostic investigation, it became clear that feed trucks were a common site of ASFV surface contamination detection. This information resulted in biosecurity‐focused actions for feed trucks arriving back at the feed mill, including decontamination of interior truck cab surfaces and washing of exterior truck surfaces with high‐pressure water prior to application of surface disinfectants. Additionally, a low number of rt‐PCR positive samples were detected within the feed production system, with the greatest number coming from transient surfaces such as high traffic areas and worker clothing. This illustrates the importance of managing employee traffic through procedures such as zoning and separation between clean–dirty areas to reduce the likelihood of pathogen transmission. In conclusion, this report illustrates the importance of routine data capture regarding efficacy of biosecurity procedures which allows for real‐time updates and improvement as biosecurity gaps are identified.


INTRODUCTION
African swine fever virus (ASFV) is a major challenge for the global swine production industry. The virus is a member of the Asfarviridae family (Galindo & Alonso, 2016), and is relatively large in size compared to many other viruses. It is a double-stranded DNA virus with a viral envelope consisting of a lipid bilayer membrane (Galindo & Alonso, 2016). To date, there is no efficacious vaccine approved and available for ASFV. Several outbreaks have been reported in recent decades, notably in Georgia in 2007 and China in 2018. Following the introduction into China in 2018, ASFV rapidly spread throughout Southeast Asia. Since ASFV was first introduced into Vietnam in February 2019, it quickly spread to all provinces and urban administrative districts within the country. Information regarding the number of animals culled due to ASFV is limited, but reports in excess of 6 million animals have been provided (FAO, 2021). Pathogens causing swine disease are known to be transmitted via a wide variety of direct and indirect pathways. This is true for ASFV, and multiple transmission pathways have been described including direct pig to pig contact, consumption of contaminated food products commonly known as swill feeding, or other fomites such as vehicles, workers, and other equipment (Bellini et al., 2016;Guinat et al., 2016;Olesen et al., 2020). Once potential pathways of disease transmission have been identified, corrective measures can be put in place to reduce the likelihood of transmission. This is the foundation of biosecurity, and routine monitoring for presence of an infectious agent can help identify potential gaps in biosecurity practices.
However, gaps in knowledge currently exist involving the epidemiology of ASFV transmission, particularly through the supply of swine feed and transportation biosecurity. Therefore, the objective of the current investigation was to monitor for the presence of ASFV within a live swine production, feed manufacturing, and distribution system located in an area of ongoing ASFV circulation with the goal of identifying biosecurity gaps so corrective measures can be implemented.

Case description
The diagnostic investigation was performed in collaboration with a production company located in Vietnam. This company owns and operates multiple feed manufacturing and live swine production facilities located in some of the most pig-dense regions of Vietnam. The collaborator is a farrow-finish producer that incorporates a high level of biosecurity and modern swine production practices including multisite production, all-in/all-out pig flow, routine decontamination of facilities, and animal transport vehicles between animal groups. As previously indicated, a complex network of transmission pathways exists for ASFV and other swine diseases. Within this investigation, the feed supply chain and live animal marketing station were the primary pathways of interest because the collaborator had the greatest ability to directly incorporate biosecurity improvements to these areas if gaps were identified. The feed supply chain pathway includes a feed mill and associ-ated ingredients, finished feed, surfaces, as well as vehicles for delivery of ingredients and finished feed. Additionally, the production system raises and markets pigs to buyers via a company-owned market pig transfer station. This facility as well as live market animal transportation vehicles was the second key possible ASFV transmission pathway of interest for this investigation.
Feed for this production system is manufactured by companyowned feed mills. These feed mills also manufacture and distribute feed to customers outside of their own live animal production. In brief, Fixed location surfaces in non-production airspace with potential of exposure through contact with fomites OR non-fixed location surfaces with intermittent contact with surfaces with potential prior exposure to suspected pathogen transmission entity.

Sample collection
Throughout the investigation, a variety of samples were collected gauze sponges that were pre-moistened with 5 ml phosphate-buffered saline (PBS) and placed in a clean sealable plastic bag. For each swab sample, the sampling technician used a new sterile glove when handling the swab. At the pre-determined sampling location, the pre-moistened swab was used to sample an area approximately 20 × 20 cm 2 using a crossing pattern where the surface is swabbed vertically to cover area, and then is swabbed horizontally over the same area to ensure appropriate coverage. The swab sample was then placed back in the sealable plastic bag for transportation to the diagnostic laboratory.

Sample analysis
The diagnostic laboratory was owned and operated by the collaborator and was established to perform routine diagnostic testing for the live animal production enterprise. All samples were processed and analyzed locally, and no material whatsoever was transported out of the region. For analysis of feed and ingredient samples, the sampling bag containing the feed or ingredient sample was manually agitated to ensure an even mix, and then the bag was opened aseptically to collect a representative subsample of 5 g of material. The 5-g subsample was then placed in a sterile container and 25 ml of PBS was added, agitated for 1 h, and the supernatant liquid was collected for real-time polymerase chain reaction (rt-PCR) analysis. Environmental swabs were processed by adding 20 ml of PBS to the swab, lightly agitated, and the liquid was collected for rt-PCR analysis. If samples were not able to be analyzed within approximately 24 h due to high laboratory submission load, samples were placed in −80 • C freezer until time of analysis.
All samples were analyzed using a commercially available rt-PCR assay (VDx ASFV qPCR; Median Diagnostics Inc., Chuncheon, South Korea) designed to detect the p72 viral genome of ASFV following manufacturer recommendations. Amplification was performed using a Stratagene MX3005P qPCR system (Aligent Technologies, Waldbronn, Germany). A total of 40 PCR amplification cycles were used, and if any sample did have detectable ASFV DNA it was considered rt-PCR positive. If a sample did contain ASFV DNA, values are reported as cycle threshold (Ct).

Statistical analysis
Data were analyzed using linear models using the GLIMMIX procedure in SAS 9.4 (SAS Institute Inc., Cary, NC, USA). Data were categorized in a binary manner based on rt-PCR result (0 if no detected ASFV DNA, 1 if ASFV DNA detected) and fit using a binary distribution, logit link, Laplace approximation, and ridge-stabilized Newton-Raphson algorithm as described by Magossi, Bai, et al. (2019) and . Fixed effects in the statistical models included sampling zone, sampling month, and the associated interaction. For all models, the interaction term was not significant (P > .10) and therefore was excluded from the final model. For each sampling location, the overall percentage of samples that were rt-PCR positive was calculated using the FREQ procedure. Mean probabilities for rt-PCR positive status are reported along with 95% confidence limits.

Feed delivery vehicles
As an initial assessment of the potential detection rate of ASFV DNA, the feed supply chain was selected as the sampling location of importance due to the highly connected structure among farms. For all sampling locations, there was no evidence of sampling zone × month interaction (P ≥ .986). During the first set of samples collected (78 samples in August 2019 from feed mill environment, feed ingredients, and feed delivery trucks), there were four samples with detected ASFV contamination (Table 2). All four samples were from the cabs of feed delivery trucks (Table 3)

Feed mill environment
There was no evidence of a difference in detection of ASFV contamination between feed contact, non-feed contact either less than or greater than 1 m from feed contact surfaces, or transient surfaces (P = .660).
Overall, nine total environmental samples were rt-PCR positive within the feed mill (total of 1100 total samples; percent positive within sampling zone ranging from 0.40% to 1.22%; Table 4). There also was no evidence that detection of ASFV differed by month (P = .983). Within the feed mill, the first rt-PCR positive sample was found in September 2019 from a high-traffic surface where truck drivers congregate once returning to the mill. The first feed contact surface with evidence of ASFV contamination was in February 2020, and was followed by two additional rt-PCR positive samples on feed contact surfaces in April 2020.
Worker clothing was identified as a source of contamination in March TA B L E 2 Real-time polymerase chain reaction (rt-PCR) analysis of environmental swabs collected from feed delivery vehicle surfaces collected in a region of active African swine fever virus (ASFV) circulation a

Ingredients and finished feed
Overall, only one rt-PCR positive sample out of 142 samples collected from finished feed, ingredients, or water used in feed processing was found ( Table 5). The total number of feed and ingredient samples collected in the current investigation was limited, as it was believed that environmental samples would be more reflective of contamination rate over a longer period of time compared to a point-in-time sample of feed or ingredient.

Market pig transfer station
In addition to the feed manufacture and distribution system, the collab-   Of the 22 rt-PCR positive samples, 18 were from live animal transport vehicles ( Table 6). Eight of the rt-PCR positive samples were from animal contact surfaces within the cargo hold or from wheels, whereas 10 rt-PCR positive samples were collected from within the truck cabs.
Only one rt-PCR positive sample was collected from an animal contact TA B L E 6 Real-time polymerase-chain reaction (rt-PCR) analysis of environmental swabs collected from live animal transport vehicles at a market pig transfer facility in a region of active African swine fever virus (ASFV) circulation a

DISCUSSION
African swine fever is a major challenge for the global swine industry with serious economic implications not only due to the impact on animal morbidity and mortality, but also the wide-ranging implications on global trade. ASFV is the sole member of the family Asfarviridae, and is a DNA virus very stable in certain environments such as meat products (Galindo & Alonso, 2016;Gaudreault et al., 2020). Transmission of ASFV has been documented to occur through a wide variety of pathways including direct pig to pig contact, consumption of contaminated food products commonly known as swill feeding, or other indirect forms of transmission such as via vehicles, workers, and other equipment (Bellini et al., 2016;Guinat et al., 2016;Olesen et al., 2020).
Additionally, ASFV is unique due to the presence of a sylvatic transmission cycle via the soft tick genus Ornithodoros (Galindo & Alonso, 2016). Because of the high degree of viral stability in a number of conditions and wide variety of transmission pathways, control of ASFV is very challenging with historically very limited eradication success.
In the current investigation, our goal was to understand the potential areas within a swine live production, feed manufacturing, and distribution system where detection of ASFV DNA could be found. This sampling approach allows for diagnostic information to be gathered and used to make biosecurity improvements. The swine feed supply is a highly linked system, with one feed mill often serving a large number of farms oftentimes over great geographic distances. Consumption TA B L E 7 Real-time polymerase-chain reaction (rt-PCR) analysis of environmental swabs collected from surfaces within market pig transfer facility in a region of active African swine fever virus (ASFV) circulation a  sample an area approximately 20 × 20 cm 2 , then 20 ml of phosphate-buffered saline was added, lightly agitated, and liquid extracted for ASFV p72 rt-PCR analysis. b Model-adjusted percentage of samples rt-PCR positive with 95% confidence limits. Sampling zone × month interaction, P = 1.000. c Zones defined as: zone 1 = animal contact surface; 3 = non-pig contact surface; 4 = transient surface.
of contaminated swine feed has been documented to be a potential route of transmission for a number of diseases including porcine epidemic diarrhoea virus (PEDV; Aubry et al., 2017;Bowman et al., 2015) and ASFV (Niederwerder et al., 2019). In addition to consumption of contaminated feed, the potential for indirect transmission via fomites associated with feed manufacture and delivery (personnel, vehicles, equipment) serves as an additional source of potential pathogen transmission. Because of the high degree of interconnectedness of the feed supply, the current investigation began by evaluating surfaces associated with the manufacture and delivery of swine feed. Additional information reviewing the principles underlying biosecurity plans for feed mills and review of strategies to reduce pathogen transmission through swine feed can be found elsewhere (Cochrane et al., 2016;Stewart et al., 2020).
Early in the investigation, it was identified that feed delivery truck cabs were a common source of ASFV DNA detection. Although this finding was eye opening to the researchers and production system, it is quite logical given the high degree of personnel movement into and out of these areas and relative difficulty to clean and decontaminate compared to exterior vehicle surfaces. Similar results were observed by Greiner (2016) who found that truck foot pedals were the most common areas within a feed mill where contamination of PEDV or porcine deltacoronavirus could be detected. Furthermore, with ASFV specifically, risk-based modelling has demonstrated that trucks traveling from regions with ASFV circulation are a potential route of disease introduction into a previously naïve region (Mur et al., 2012). As a result of the detection of ASFV DNA within truck cabs, the production system changed their biosecurity practices and incorporated a liquid disinfection procedure where disinfectant was aerosolized using a portable blower with a mounted tank and application nozzle and directed into the truck cabs upon returning to the feed mill prior to entering the perimeter boundary. The system reviewed procedures for employee traffic associated with the delivery of feed and modified procedures to reduce risk of surface contamination if the farm was in a pre-clinical stage of disease by instituting a policy where delivery truck drivers do not exit the vehicle. Additionally, the production system incorporated a washing system with high-pressure water for the exterior surfaces of all feed delivery trucks returning to the feed mill prior to application of the disinfectant mist. These changes in biosecurity procedures illustrate that when diagnostic sampling is planned and executed in a strategic manner, the information generated can be extremely valuable and can help generate improvements in biosecurity.
In addition to the information widely available regarding ASFV stability in meat products over extended periods of time, ASFV DNA has been detected in feed ingredients including dried porcine blood (Wen et al., 2019). Additional research has indicated many of these pathogens can extend for significant periods of time and even remain viable after transoceanic shipment (Dee et al., 2018). Furthermore, it has been documented that pigs can be experimentally infected with ASFV when delivered either via drinking water or via swine feed (Niederwerder et al., 2019). In the current investigation, we focused our sampling efforts on environmental samples from within and immediately surrounding the feed mill. Based on previous research with PEDV, once introduced into a feed manufacturing facility, PEDV becomes widely distributed throughout the facility and can be readily detected through environmental sampling .
Additionally, the ability to detect viral RNA within the environment can persist for extended periods of time, and can only be removed through intensive cleaning, disinfection, and thermal processing measures . Recently, evidence has been generated that indicates ASFV can become widely distributed within a feed mill when manufacturing inoculated feed in a controlled setting (Elijah et al., 2021). Additional research is necessary to further understand these environmental distribution characteristics in a real-world setting, but initial evidence indicates that environmental sampling for ASFV within a feed manufacturing facility may be a useful diagnostic tool similar to sampling for PEDV.
Of the 142 finished feed, ingredient, and water samples collected from the feed mill, only one contained detectable ASFV DNA. This is consistent with recent results reported by Yan et al. (2020), which indicated ASFV PCR-positive rates ranging from 0.2% to 1.8% in pooled complete feed and ingredient samples across three feed mills in China.
Additionally, these data are in agreement with risk assessments put forth by the United States (USDA, 2019) and European Union (EFSA, 2020), which largely describe a low risk of ASFV entry through plantbased feeds and ingredients. This experiment reveals a directionality of contamination most likely originated from infected farms and transferred via personnel and vehicles. ASFV is considered endemic in Vietnam, which has a variety of ingredient growing and processing condi- In the current investigation, several key areas of environmental contamination were identified including high-traffic areas and worker clothing. Likewise, the greatest area for contamination within the live animal transfer infrastructure sampled was live animal transport vehicles, specifically the cabs of these vehicles. When evaluating the semiquantitative Ct values, there are very high Ct values near the limit of detection for rt-PCR assays therefore repeatability is a concern. We acknowledge these assays were developed to detect ASFV DNA in biological tissues of infected animals. However, the detection noted in this study indicates the need for development of assay methods that have lower and more consistent detection limits. In general, these data illustrate that surface contamination with ASFV is possible within a region of ongoing transmission.
The stability of ASFV in the environment is somewhat unknown.
There have been reports that environmental survival is relatively short (Olesen et al., 2018), although other field reports indicate a much longer duration of potential infectivity especially in the presence of organic material (Davies et al., 2017;Fischer et al., 2020;Mazur-Panasiuk & Wozniakowski, 2020 It is important to consider limitations of the current investigation.
One limitation is that there was no measure of sample infectivity.
Detection of ASFV DNA via rt-PCR gives no indication that a sample is infectious. Therefore, it is not possible to infer the likelihood of infection in a naïve population of pigs if either direct or indirect contact is made. There are several advantages of using rt-PCR compared to other diagnostic assays including the flexible sample types which can be analyzed and rapid turnaround time (Rodriguez et al., 2009). It is possible, however, and we believe extremely useful to use this information to identify potential gaps in biosecurity and implement corrective measures to reduce detection of ASFV DNA. Additionally, it is difficult to ascertain whether the low number of PCR positive samples is due to a true low prevalence of contamination, or issues with sample collection, processing, and molecular diagnostic techniques for these challenging sample types. There is additional information to be learned regarding the challenges associated with extraction of viral genetic material from challenging sample types such as feed and environmental samples, but we believe the current investigation highlights how these methods can be incorporated in the field to aid with biosecurity practice assessment.
The use of rt-PCR tests can serve as a rapid, cost-effective method to evaluate the efficacy of biosecurity practices and identify potential areas for improvement.
Another limitation of the current study is the nature of the investigation was to identify biosecurity gaps with the goal of implementing corrective improvements in biosecurity. Therefore, the biosecurity practices of the system were adjusted as more data were generated and these data are not true estimates of prevalence of ASFV DNA detection within this system over time. It is also important to consider that changes in ASFV DNA detection over time could be attributed to biosecurity improvements, but also could be due to changes in disease prevalence within the region. This system also incorporated a high level of biosecurity within many areas of live animal production as well as feed manufacture and delivery, which means extrapolation to other situations may not be appropriate or provide an accurate assessment of risk. The feed mill in the current investigation also pellets all diets and uses a commercially available formaldehyde-based feed product in a subset of manufactured diets. Therefore, it is not possible to extrapolate these results beyond these particular conditions, and the rate of ASFV DNA detection may differ under different conditions. This investigation has illustrated the value that environmental sampling can bring to a system's biosecurity assessment and protocol development. Environmental sampling can also serve as a method of indicating the source of a disease outbreak and its directionality through fomite transmission. Most feed safety research is based on the theory of forward transmission, with a contaminated ingredient being mixed into feed and causing an initial outbreak in animals. However, the evidence in this case instead suggests the possibility of backward contamination, where an outbreak on the farm led to contamination of feed trucks which transferred the contamination back to the feed mill.
In summary, a wide variety of factors must be considered to reduce the likelihood of disease transmission and this information provides baseline understanding in a real-world setting which can be used as a foundation for future investigations to build upon.